The processing of soluble protein antigens to peptide fragments by antigen-presenting cells (APCs) is usually required for the stimulation of CD4+ T cells. The mechanism and location whereby APCs produce antigenic peptides are unclear. The major objective of this research proposal is to define the mechanism of antigen processing by determining the involvement of particular intracellular transport pathways and acidic compartments. This objective will be accomplished by targeting antigens along selected transport pathways. By a heteroligation technique, antigens will be conjugated to transferrin and alpha 2-macroglobulin which enter cells by receptor-mediated endocytosis. In receptor bearing cells, transferrin conjugates should be directed away from the lysosomes, whereas alpha 2-macroglobulin conjugates should be delivered to the lysosomes. Conjugates will be isolated by gel filtration, and their composition will be assessed by polyacrylamide gel electrophoresis. First, the role of these pathways in antigen processing will be determined by the ability of receptor positive APCs to process the conjugates resulting in stimulation of antigen-specific CD4+ T cell clones to hydrolyze phosphatidylinositol 4,5 bisphosphate in a serum-free assay. This response will be compared to that induced by receptor negative APCs which should internalize the conjugates by fluid-phase pinocytosis. The exact pathway by which the conjugates travel inside receptor positive APCs will be determined by the location of radiolabeled conjugates over time in subcellular fractions isolated by Percoll density centrifugation and compared with free transferrin and alpha 2- macroglobulin. Second, the requirement of endosomal acidification in processing the conjugates will be determined by the use of temperature sensitive mutant APCs which lose endosomal, but not lysosomal, acidification at the higher temperature. The ability of the mutant cells, which already express the appropriate receptors, to process the conjugates at both temperatures will be compared to that of wildtype parental cells. Third, the immunological relevance of these pathways will be determined by the ability of the conjugates to elicit an immune response in vivo. After administration of the conjugates to mice, the specificities of a panel of generated CD4+ T cell hybridomas will be analyzed by the use of fragments of the conjugates. This research proposal will increase the understanding of the role of APCs in a CD4+ T cell response. Knowledge in this area is important for future studies on the pathogenesis of autoimmune disease and development of vaccines.